Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
  • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/174—Vitamins

Definitions

• the present invention relates to an improved process for the preparation of D-pantothenic acid and / or its salts and the use as an additive to animal feed.
• D-pantothenate As a starting product for the biosynthesis of coenzyme A, D-pantothenate is widely used in plants and animals. In contrast to humans, who ingest sufficient amounts of pantothenic acid through food, deficiency symptoms for D-pantothenate are frequently described for both plants and animals. The availability of D-pantothenate is therefore of great economic interest, especially in the animal feed industry.
• D-pantothe ⁇ ate is conventionally prepared by chemical synthesis from D-pantolactone and calcium- ⁇ -alaninate (Ullmann's Encyclopedia of Industrial Chemistry, 6 01 edition, 1999, electronic release, chapter "Vitamins").
• D-pantolactone The complex sales product resulting from chemical synthesis is usually the calcium salt of D-pantothenic acid, calcium D-pantothenate.
• the advantage of biotechnological manufacturing processes with microorganisms lies in the selective (enantiomerically pure) provision of the D-form of pantothenic acid that can be used by the higher organism.
• a complex resolution of racemates, as is required in chemical synthesis, is thus eliminated.
• Fermentative processes for the production of D-pantothenic acid with microorganisms are well known, e.g. from EP 0 590 857, WO 96/33283, US 6,013,492, WO 97/10340, DE 19846 499, EP 1 001 027, EP 1 006 189, EP 1 006 192 and EP 1 006 193.
• EP 1 006 189 and EP 1 001 027 describe processes for the production of Pantothenate in which a content of at most 1 g / l D-pantothenic acid is reached in the fermentation solution.
• Such low pantothenic acid contents in the fermentation solution ie less than 10% by weight based on the solids content, are unsuitable for the economical production of animal feed supplements containing D-pantothenic acid.
• Another disadvantage of the processes described so far is that the isolation of the product from the fermentation medium requires numerous complex work-up steps. An economical manufacturing process for the industrial scale is not disclosed.
• the published patent application DE 100 16 321 describes a fermentation process for the production of an animal feed supplement containing D-pantothenic acid.
• a major disadvantage of this process is that the pantothenic acid precursor ⁇ -alanine must be added to the microorganism via the fermentation medium in order to obtain economic yields of the desired product.
• US Pat. No. 6,013,492 and WO 96/332839 describe working up the D-pantothenic acid from the fermentation solution by filtering off insoluble constituents (for example cell material) from the culture medium, adsorbing the filtrate on activated carbon, then eluting the D-pantothenic acid with an organic one Solvent, preferably methanol, neutralization with calcium hydroxide and a final crystallization of calcium D-pantothenate.
• organic one Solvent preferably methanol
• EP 0 590 857 describes a fermentation process for the production of D-pantothenic acid, in which the cultivation of a microorganism absolutely requires the addition of ⁇ -alanine.
• the fermentation solution is filtered to separate the biomass, then passed through a cation exchanger and then through an anion exchanger, then neutralized with calcium hydroxide, evaporated, mixed with activated carbon, filtered again and crystallized with the addition of methanol and calcium chloride.
• the resulting calcium pantothenate-containing product contains, in addition to D-pantothenic acid in the form of the calcium salt, calcium chloride in a molar ratio of 1: 1.
• Electrodialysis followed by spray drying is required to reduce the calcium chloride content.
• the disadvantage of this process is that it is neither economical nor ecological because of the large number of complex process steps and the use of organic solvents.
• the object of the present invention is to provide an animal feed supplement containing D-pantothenic acid and / or its salts, and the production thereof by an improved process for producing D-pantothenic acid and / or its salts which does not have the disadvantages mentioned above.
• a process is desirable in which the addition of ⁇ -alanine is drastically reduced or is not required at all.
• the production of D-pantothenic acid in the form of its divalent salts, and especially the alkaline earth metal salts is desirable since the divalent salts have less hygroscopic properties than monovalent salts of pantothenic acid and for further use, e.g. B. as an animal feed supplement, thus less prone to clumping.
• the present invention relates to a process for the preparation of D-pantothenic acid and / or its salts, characterized in that a) at least one organism producing D-pantothenic acid is used, the pantothenic acid (pan) and / or isoleucine valine (ilv ) - Biosynthesis is deregulated and forms at least 2 g / l of salts of D-pantothenic acid by fermentation in a culture medium, 0-20 g / l of free ß-alanine and / or ß-alanine salt being added to the culture medium, b ) the fermentation solution containing D-pantothenate is passed over an anion exchanger, c) the D-pantothenate bound to the anion exchanger with a solution containing inorganic or organic calcium and / or magnesium salts in the form of calcium and / or magnesium D-pantothenate or with an HCl solution in the form of free D-pantothenic acid is eluted, d)
• Calcium and / or magnesium base is adjusted to a pH of 3-10, a solution is obtained which contains calcium and / or magnesium pantothenate and e) the eluate or the solution containing calcium and / or magnesium Pantothenate is subjected to drying and / or formulation.
• the present method is characterized in that in step d) or in step e) a suspension is obtained or presented which contains calcium and / or magnesium pantothenate.
• step a) The fermentation which takes place in step a) can be carried out according to procedures known per se in batch, fed-batch or repeated fed-batch operation or with continuous process control.
• customary buffer systems such as, for. B. phosphate buffer with NaOH, KOH or ammonia.
• step a) at least 10 g / l, preferably at least 20 g / l, particularly preferably at least 40 g / l, most particularly preferably at least 60 g / l and in particular at least 70 g / l of salts of D-pantothenic acid formed by fermentation in the culture medium.
• the expression “produce” means that the organism can synthesize larger amounts of D-pantothenic acid and / or its salts than are required for its own metabolic needs.
• the amount of D-pantothenic acid and / or their salts do not pre-exist inside the cell, but are ideally completely released from the organism into the culture medium, which can be actively or passively discharged according to mechanisms known per se.
• microorganisms are used as the organisms producing D-pantothenic acid. According to the invention, these include fungi, yeasts and / or bacteria. According to the invention, fungi such as, for example, Mucor or yeasts, such as. B.
• coryneform bacteria or Bacillaceae are advantageously used.
• B. pantothenticus B. circulans
• B. coagulans are particularly preferred here, for example B. megaterium, B. pumilus, B. thuringiensis, B. brevis, B. stearothermophilus and other group 1 Bacillus species characterized by their 16sRNA or Actinum mycetalis. This list is for the purpose of explanation and is in no way limitative of the present invention.
• the present invention also includes the use of genetically modified organisms for the production according to the invention of an animal feed supplement containing free D-pantothenic acid and / or its salts.
• genetically modified organisms can be isolated, for example, by chemical mutagenesis and subsequent selection by means of a suitable “screening method”.
• so-called “production strains” are also included which are suitable for the production of the product in the sense of the present invention and have genetic changes with regard to the metabolic flow in the direction D-pantothenic acid, which also includes changes in the discharge of D-pantothenic acid and / or its salts across the cell membrane. This can e.g. B. by changes in key positions in relevant metabolic biosynthetic pathways of the organism used.
• transgenic organisms which result from the transfer of homologous and / or heterologous nucleotide sequences which lead to Synthesis of the desired product are necessary or can be beneficial.
• the overexpression and / or deregulation of one or more genes individually and / or in combination, localized in the genome and / or on a vector is conceivable.
• Such transgenic organisms can advantageously contain additional copies and / or genetically modified genes selected from the group of panB, panC, panD, panE and / or their combinations and / or even organizational units, such as the panBCD operon.
• metabolic pathways such as the isoleucine-valine biosynthetic pathway in the organisms
• branched-chain precursors of pantothenic acid biosynthesis are increasingly being made available.
• the genes for this biosynthetic pathway ie ilvB, ilvN, ilvC and / or ilvD, are advantageously overexpressed.
• genetic changes in the aspartate ⁇ -decarboxylase (panD) for example by overexpression and / or deregulation, in the organism producing D-pantothenic acid used are included according to the invention.
• the term “deregulation” means the following: Change or modification of at least one gene which codes for an enzyme in a biosynthetic pathway, so that the activity of the enzyme in the microorganism is changed or modified. It is preferred that at least one gene which codes for an enzyme of a biosynthetic pathway is modified in such a way that the gene product is produced more or has an increased activity.
• the term “deregulated pathway” also includes a biosynthetic pathway in which more than one gene is responsible for more encoded as an enzyme is modified or modified so that the activities of more than one enzyme are changed or modified.
• Changes or modifications may include, but are not limited to: removing the endogenous promoter or regulatory elements; Inserting strong promoters, inducible promoters, or multiple promoters simultaneously; Removing regulatory sequences so that the expression of the gene product is changed; Change in the chromosomal location of the gene; Changing the DNA sequence near the gene or within the gene like z.
• Deregulation can also involve changes in the coding region of genes, e.g. B. lead to cancellation of feedback regulation in the gene product or to a greater or lesser specific activity of the gene product.
• genes encoding such enzymes are: alsD, avtA, ilvE, ansB, coaA, coaX and others.
• Enumeration serves as an explanation and is in no way limiting for the present one
• ß-alanine is already present in the cells in increased concentrations compared to correspondingly non-genetically modified organisms and thus does not have to be added to the culture medium as a precursor, as is the case, for. B. in EP-A-0 590 857 is required.
• Microorganisms whose pantothenic acid (pan) and / or isoleucine valine (ilv) biosynthesis and / or aspartic ⁇ -decarboxylase (panD) are deregulated are advantageous.
• An additional overexpression of ketopanthoat reductase (panE) in the microorganisms is also advantageous.
• coaA gene which is required for the synthesis of coenzyme A is reduced or if it is completely switched off (for example in Bacillus species).
• coaX another gene for this enzymatic function
• the activity of this gene coaX or of the corresponding enzyme can also be changed, preferably reduced, or even deleted, provided that coaA itself still has sufficient, albeit reduced, enzyme activity, ie the enzyme activity of coaA has not been completely lost.
• genetic manipulation of the promoter regions of these genes is advantageous in such a way that this manipulation leads to overexpression of the gene products.
• the bacterial strains described in accordance with the appendix such as Bacillus subtilis PA 824 and / or derivatives thereof.
• the microorganism Bacillus subtilis PA 668 as described in accordance with the appendix (US Serial No. 60 / 262,995), is used in the method according to the invention.
• the strain PY79 was generated by transduction of the Trp + marker (from the Bacillus subtilis wild type W23) classical genetic engineering methods (as described, for example, in Harwood, CR and Cutting, SM (editors), Molecular Biological Methods for Bacillus (1990) John Wiley & Sons, Ltd., Chichester, England), introduced ⁇ panB and ⁇ panEI mutations
• the strain was transformed with genomic DNA from Bacillus subtilis strain PA221 (genotype P 26 panßCD, t ⁇ C2 (Trp " )) and genomic DNA from Bacillus subtilis strain PA303 (genotype P 2 ⁇ panE1).
• the resulting strain PA327 has the genotype Pi anBCD, P 2 ⁇ anE1 and is tryptophan auxotroph (Trp).
• Bacillus subtilis strain PA327 was made up to 740 in 10 ml cultures with SVY medium (25 g / L Difco Veal Infusion Broth, 5 g / L Difco Yeast Extract, 5 g / L Na glutamate, 2.7 g / L ammonium sulfate Fill up with water, autoclave, then add 200 ml of 1 M potassium phosphate, pH 7.0 and 60 ml of 50% sterile glucose solution), which was supplemented with 5 g / L ß-alanine and 5 g / L ⁇ -ketoisovalerate, pantothenic acid Titer of up to 3.0 g / L (24 h) reached.
• the preparation of the Bacillus subtilis strain PA221 (genotype P 2 epanBCD, t ⁇ C2 (T
• the panBCD operon from Bacillus was derived from a Bacillus subtilis GP275 plasmid library using classical genetic engineering methods and the sequence information of the panBCD operon from E. coli (see Merkel et al., FEMS Microbiol. Lett, 143, 1996: 247-252) cloned.
• E. coli strain BM4062 Bir te
• the panBCD operon was introduced into a plasmid replicable in E. coli.
• This plasmid was transformed into the Bacillus subtilis strain RL-1 (derivative of Bacillus subtilis 168 (Marburg strain ATCC 6051) obtained by classic mutagenesis, genotype trpC2 (Trp " )) and the native panBCD operon was transformed by the p 2 6panBCD by homologous recombination
• the resulting strain is called PA221 and has the genotype P 26 panßCD, £ ⁇ C2 (Trp " ).
• Bacillus subtilis strain PA221 was used in 10 ml cultures with SVY medium supplemented with 5 g / L ß-alanine and 5 g / L ⁇ -ketoisovalerate, pantothenic acid titer of up to 0.92 g / L (24 h) reached.
• panE1 and panE2 The Bacillus panE sequence was cloned analogously using the E. coli panE gene sequence. It turned out that in ß. subtilis two homologues of the panE gene from E. coli exist, which were designated panE1 and panE2. Deletion analyzes showed that the panE1 gene is responsible for 90% of pantothenic acid production, while the deletion of the panE2 gene had no significant effect on pantothenic acid production.
• the promoter was replaced by the strong constitutive promoter P 26 and the ribosome binding site in front of the panE1 gene was replaced by the artificial binding site.
• the P 25 panE1 fragment was cloned into a vector that was designed so that the P 2 epanE1 fragment could integrate into the original panE1 locus in the genome of Bacillus subtilis.
• the strain resulting after transformation and homologous recombination is called PA303 and has the genotype P 2e panE1.
• Bacillus subtilis strain PA303 was used in 10 ml cultures with SVY medium supplemented with 5 g / L ß-alanine and 5 g / L ⁇ -ketoisovalerate to produce pantothenic acid titers of up to 1.66 g / L (24 h) reached.
• the further trunk construction was carried out by transforming PA327 with a
• P 26 panE1, P 26 // vß ⁇ / C, specR, trpC2 (Trp ' )).
• pantothenic acid titer of up to 3.6 g / L (24 h) was achieved in 10 ml cultures with SVY medium supplemented only with 5 g / L ß-alanine, in 10 ml Cultures with SVY medium supplemented with 5 g / L ß-alanine and 5 g / L ⁇ -ketoisovalerate achieved pantothenic acid titres of up to 4.1 g / L (24 h).
• a deregulated / YvD cassette was also introduced into the strain PA340.
• P contains 26 promoters with the artificial RBS2, transformed into PA340.
• the P 2 eilvD gene was integrated into the original ilvD locus by homologous recombination.
• the resulting strain PA374 has the genotype P 26 pat? SsCD, P anE1, P 26 ilvBNC,
• the Bacillus subtilis strain PA374 was used in 10 ml cultures with SVY medium supplemented only with 5 g / L ß-alanine to produce pantothenic acid titers of up to 2. 99 g / L (24 h) reached.
• pantothenic acid titers of up to 1.31 g / L (24 h) were achieved in 10 ml cultures with SVY medium precursor-free feed.
• Bacillus subtilis strain PA401 (genotype P panD) is described in the following section:
• the Bacillus subtilis panD gene was cloned from the panBCD operon into a vector that carries the tetracycline marker gene.
• the promoter P 26 and an artificial RBS described above were cloned in front of the panD gene.
• a fragment containing the tetracycline marker gene and the P 2 ⁇ anD gene was produced by restriction digestion. This fragment was religated and transformed into the strain PA221 described above. The fragment integrated into the genome of the PA211 strain.
• the resulting strain PA401 has the genotype P 2 epanBCD, P 2 ⁇ anD, tetR and rpC2 (Trp).
• pantothenic acid titer of up to 0.3 g / L (24 h) was achieved in 10 ml cultures in SVY medium supplemented with 5 g / L ⁇ -ketoisovalerate.
• pantothenic acid titers of up to 2.2 g / L (24 h) were achieved.
• strain PA377 a tryptophan prototrophic strain was generated by transformation with chromosomal DNA from strain PY79.
• This strain PA824 has the genotype P 2 ⁇ panBCD, P 2 ⁇ anE1, P 26 / 7vß / VC, P 26 / 7vD, specR, tetR and Trp + .
• Bacillus subtilis strain PA824 was used in 10 ml cultures in SVY medium to pre-feed-free pantothenic acid titer of up to 4.9 g / L (48 h) (comparison PA377: up to 3.6 g / L in 48 h) reached.
• PA668 The production of PA668 is described in the following section:
• the Bacillus panB gene was cloned from the wild-type panBCD operon and inserted into a vector which, in addition to a chloramphenicol resistance gene, also ß. contains subtilis sequences of the vpr locus.
• the strong constitutive promoter P 2 6 was before the 5 'end of the panB gene introduced.
• a fragment containing the P 2 % panB gene, the marker gene for chloramphenicol resistance and Bacillus subtilis vpr sequences was obtained by restriction digestion. The isolated fragment was religated and the strain PA824 was transformed with it. The strain obtained was designated PA668.
• the genotype of PA668 is: P 2 ⁇ anBCD, P 2 ⁇ anE1, P 2 & ilvBNC, P ⁇ ilvD, P 2 ⁇ anB, specR, tetR, CmR and Trp + .
• PA668-2A Two colonies of PA668 were isolated and designated PA668-2A, the other PA668-24.
• PA668-2A With ß. subtilis strain PA668-2A are grown in 10mL cultures in SVY medium without
• pantothenic acid titer Feeding of precursors pantothenic acid titer of 1.5 g / L achieved in 48 h. In 10 mL cultures supplemented with 10g / L aspartate, titres up to 5 g / L are achieved.
• strain PA377 is used for glucose-limited fermentation in SVY medium (25 g / L Difco Veal Infusion Broth, 5 g / L Difco Yeast Extract, 5 g / L tryptophan, 5 g / L Na glutamate, 2 g / L (NH 4 ) 2 SO 4 , 10 g / L KH 2 PO 4 , 20 g / LK 2 HPO, 0.1 g / L CaCI 2 , 1 g / L MgSO 4 , 1 g / L sodium citrate, 0, 01 g / L FeSO 4 x7 H 2 O and 1 ml / L of a trace salt solution with the following composition: 0.15 g Na 2 MoO x 2 H 2 O, 2.5 g H 3 BO 3 , 0.7 g CoCI 2 x 6 H 2 O, 0.25 g CuSO x 5 H 2 O, 1.6 g MnCI 2 x 4 H 2 O, 0.3 g Zn
• strain PA824 is used for glucose-limited fermentation in a medium consisting of 10 g / L Difco Yeast Extract, 10 g / L NZ Amine A (Quest International GmbH, Erftstadt), 10 g / L Na-Glutamate, 4 g / L (NH 4 ) 2 SO 4 , 10 g / L KH 2 PO, 20 g / LK 2 HPO 4 , 0.1 g / L CaCI 2 , 1 g / L MgSO 4 , 1 g / L sodium citrate, 0, 01 g / L FeSO 4 x7 H 2 O and 1 ml / L of the trace salt solution described above on a 10 L scale with continuous addition of a glucose solution in 36 h (48 h) pantothenic acid concentrations of 37 g / L (48 g / L) in Fermentation broths reached.
• a medium consisting of 10 g / L Difco Yeast Extract, 10 g / L N
• pantothenic acid concentration in the fermentation broth is conceivable by further media optimization, by increasing the fermentation time, by improving the process and strain, and by combinations of the individual steps.
• the pantothenic acid concentrations described above can also be achieved by fermentation of strains which are derivatives of the PA824 described above. Derivatives can be produced through classic strain development as well as through further genetic engineering manipulations. Through media, strain and fermentation process development, the pantothenic acid titers in the fermentation broths can be increased to over 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, and> 90 g / L.
• An important advantage of the process according to the invention is that the fermentation is carried out in a culture medium which, apart from at least one carbon and nitrogen source, contains no further precursors as starting compounds. That the biosynthesis of D-pantothenic acid is independent of the feeding of further precursors.
• such precursors include substances such as to understand ⁇ -alanine and / or L-aspartate and / or L-valine and / or ⁇ -ketoisovalerate and / or combinations thereof.
• the fermentation of the D-pantothenic acid-producing organism is carried out in a culture medium which contains a carbon and a nitrogen source, but to which no free ß-alanine and / or ß-alanine salts have been added or in Course of the fermentation is supplied.
• D-pantothenic acid in ranges of at least 10 g / l culture medium, preferably of at least 20 g / l, particularly preferably of at least 40 g / l, most particularly preferably of at least 60 g / l and in particular of at least 70 g / l
• no addition of free ß-alanine and / or ß-alanine salts is required.
• the addition of ß-alanine and / or ß-alanine salts is not excluded according to the invention, so that consequently the yield of D-pantothenic acid can be further improved by the addition of ß-alanine and / or ß-alanine salts. If, for example, it is assumed that all necessary precursors of pantothenic acid are present in sufficient quantity, only the activity of the panD gene limits a further increase in pantothenic acid production, the yield of pantothenic acid can e.g. can be increased by a further 50% by adding free ß-alanine and / or ß-alanine salts.
• up to 20 g / l of free .beta.-alanine and / or .beta.-alanine salts can be added to the culture medium to further increase the pantothenic acid yield by more than 50%. It is preferred to add about 15 g / l of free ⁇ -alanine and / or ⁇ -alanine salts to the culture medium.
• carbon sources suitable according to the invention for use in a culture medium for fermentation of the aforementioned organisms are sugars, such as starch hydrolysates (mono-, di-, oligosaccharides), preferably glucose or sucrose, and beet or cane sugar molasses, proteins, protein hydrolysates, soybean meal, corn steep liquor, fats, free fatty acids, recycled cells from fermentations already carried out or their hydrolyzates as well as yeast extract.
• starch hydrolysates mono-, di-, oligosaccharides
• glucose or sucrose preferably glucose or sucrose
• proteins, protein hydrolysates preferably soybean meal, corn steep liquor, fats, free fatty acids, recycled cells from fermentations already carried out or their hydrolyzates as well as yeast extract.
• the present process is advantageously characterized in that the total sugar content is reduced to a minimum until the end of the fermentation, since otherwise the subsequent drying and / or formulation the fermentation solution made difficult by gluing.
• This can be achieved according to the invention in that the fermentation is continued for some time after the carbon source has been used up (in cultivation in batch mode) or after the carbon supply (in process control in fed-batch or repeated-fed-batch mode) is interrupted and / or regulated in such a way that the concentration of the carbon source is almost zero (in the case of fed-batch, repeated-fed-batch or continuous process control).
• the fermentation is continued in the fermentation solution to at least 80%, preferably 90% and particularly preferably 95% of the saturation value until the dissolved oxygen concentration (pO 2 ) is reached ,
• nitrogen sources suitable according to the invention are ammonia, ammonium sulfate, urea, proteins, protein hydrolyzates or yeast extract. This list is also not limiting for the present invention.
• the fermentation medium also contains mineral salts and / or trace elements such as amino acids and vitamins.
• mineral salts and / or trace elements such as amino acids and vitamins.
• trace elements such as amino acids and vitamins.
• the organism After the fermentation medium has been inoculated with a suitable D-pantothenic acid-producing organism (with cell densities known to the person skilled in the art), the organism is cultivated, if appropriate with the addition of an anti-foaming agent.
• the pH of the medium can be regulated using various inorganic or organic alkalis or acids, e.g. NaOH, KOH, ammonia, phosphoric acid, sulfuric acid, hydrochloric acid, formic acid, succinic acid, citric acid etc.
• the D-pantothenic acid formed is in the fermentation solution in the form depending on the buffer system used of the respective salt (s). Since the salts of D-pantothenic acid in the form of their monovalent cations are particularly disadvantageous, the fermentation solution is prepared according to the invention using an anion exchanger.
• the present invention encompasses all commercially available anion exchangers. These include all strongly or weakly basic resins, preference being given to the strongly basic resins.
• the use of the following anion exchangers is advantageous according to the invention, the list only serving to explain the present invention and not restricting it: Lewatit M500, MP 500 from Bayer, Amberlite IRA 402, IRA 410, IRA 900 from Rohm & Haas, Dowex 1x2, 1x8 from Dow Chemicals, Diaion PA 306, PA 316, PA 406, PA412 or PA 418 from Mitsubishi Chemicals Corporation and Lewatit MP 62 from Bayer, MWA-1 from Dow Chemicals, Amberlite IRA 67, IRA 96SB, IRA 96 RF, Duolite A561 or A7 from Rohm & Haas, Reillex 402 from Reilly Industries, Diaion WA 21 or WA 30 from Mitsubishi Chemicals Corporation.
• the D-pantothenate and other anions are bound from the fermentation solution, while the other compounds, in particular monovalent cations, such as. As ammonium, potassium or sodium, pass through the anion exchanger and then be discarded. In this way, the undesired cations are advantageously almost completely removed from the fermentation solution.
• the D-pantothenate bound to the anion exchanger is then according to the invention with a solution containing inorganic or organic calcium and / or magnesium salts in the form of calcium and / or magnesium D-pantothenate or with an HCl solution in the form of free D- Pantothenic acid eluted.
• the invention can be acidic and / or basic inorganic and / or organic calcium and / or magnesium salts.
• the elution of the D-pantothenate bound to the anion exchanger with a solution containing acidic inorganic and / or organic calcium and / or magnesium salts is conceivable.
• the elution solution in step c) contains inorganic and / or organic calcium and / or magnesium bases.
• the basic calcium and / or magnesium salts can be included instead of or in addition to the acidic calcium and / or magnesium salts.
• the elution solution in step c) of the process according to the invention can include acidic inorganic calcium and / or magnesium salts, acidic organic calcium and / or magnesium salts, inorganic calcium and / or magnesium bases and / or organic calcium and / or magnesium bases and the like Combinations of these included.
• inorganic calcium and / or magnesium salts are advantageously to be understood as the corresponding halides. According to the invention, these are preferably CaCl 2 and / or MgCl 2 .
• Calcium hydroxide, calcium carbonate, calcium oxide, magnesium hydroxide or magnesium carbonate can be mentioned as an example of inorganic calcium and / or magnesium bases.
• Organic calcium and / or magnesium salts are understood to mean, for example, readily water-soluble salts of organic anions. These are preferably z.
• the D-pantothenate bound to the anion exchanger is eluted with a 5-10% strength CaCl 2 , MgCl 2 solution or aqueous HCl solution, the eluate being a D-pantothenate in the form of calcium or Contains magnesium D-pantothenate or as free D-pantothenic acid.
• the elution is carried out with an aqueous HCl solution, the eluate containing free D-pantothenic acid is adjusted to a pH of 3-10 by adding calcium and / or magnesium base.
• a pH value of 5-10 is advantageous.
• the solution is preferably adjusted to a pH of 5-9, particularly preferably 6-9 and very particularly preferably 6-8. In this way a solution is obtained which contains calcium and / or magnesium pantothenate.
• a suspension can also be obtained according to the invention which contains calcium and / or magnesium pantothenate.
• Calcium hydroxide, calcium carbonate, calcium oxide, magnesium hydroxide and / or basic magnesium carbonate in the form of a solid and / or as an aqueous is preferred for neutralization of the eluate containing free D-pantothenic acid Suspension added.
• the neutralization of the eluate containing free D-pantothenic acid is carried out with the aid of a calcium and / or magnesium base in the form of an aqueous suspension.
• a calcium and / or magnesium base in the form of an aqueous suspension.
• the method is characterized in that the eluate, if appropriate in step d), contains an aqueous suspension containing 2-55% by weight, preferably 10-50% by weight.
• a method is also included in which, in step d), an aqueous suspension containing 2-65% by weight, preferably 10-50% by weight and particularly preferably 20-40% by weight of calcium carbonate is added to the eluate becomes.
• the eluate is optionally in
• aqueous suspension containing 2-25% by weight, preferably 10-20% by weight, of basic magnesium carbonate is added to the eluate, if necessary in step d).
• the content of monovalent cations is determined in the manner described above by using an anion exchanger -, Potassium and / or sodium ions, reduced to a concentration of ⁇ 1g / kg eluate / solution.
• Drying and / or formulation of the eluate or the solution or suspension containing calcium and / or magnesium pantothenate is carried out according to methods known per se, such as spray drying, spray granulation, fluidized bed drying, fluidized bed granulation, drum drying or spin-flash drying (Ullmann's Encyclopedia of Industrial Chemistry, 6 th edition, 1999, electronic release, chapter "Drying of Solid Materials”).
• the gas inlet temperature at Convection drying is in the range of 100-280 ° C, preferably 120-210 ° C.
• the gas outlet temperature is 50-180 ° C, preferably 60-150 ° C. Fine particles can be separated and recycled to set a desired particle size distribution and the associated product properties. Coarse material can also be ground in a mill and then also returned.
• the method according to the invention is advantageous in the method described above to reduce complex work-up steps, in particular to dispense with the use of organic solvents, while at the same time providing a desired product with good biological value. Furthermore, according to the invention, the amount of waste water obtained is reduced significantly. This results in further savings on complex processing and disposal systems.
• the method according to the invention is advantageously characterized in that it is simpler, less prone to failure, less time-consuming, significantly less expensive and therefore more economical than conventional methods.
• At least one and / or combinations of the subsequent steps can be carried out before the drying and / or formulation of the solution or suspension, including lysis and / or killing of the biomass and / or separation of the biomass from the Fermentation solution and / or addition of additional additives and / or concentration of the fermentation solution, preferably by dehydration.
• the present invention thus also relates to a method in which the lysis and / or killing of the biomass is still carried out in the fermentation solution or only after the biomass has been separated from the fermentation solution.
• This can be done, for example, by a temperature treatment, preferably at 80-200 ° C. and / or an acid treatment, preferably with sulfuric acid or hydrochloric acid and / or enzymatically, preferably with lysozyme.
• the cells of the fermented microorganisms can be removed from the solutions or suspensions of steps a), c) or d) of the method according to the invention by filtration, separation (eg centrifuging) and / or decanting. It is also conceivable for the solution from step a) to be passed directly via an anion exchanger without the organisms contained being separated off.
• the processing step by anion exchanger (step b)) of the method according to the invention is not preceded by any separation of the biomass, the fermentation solution containing biomass can advantageously also be passed through the ion exchanger bed from bottom to top, ie against gravity.
• the solution resulting from the work-up via the anion exchanger can, optionally after the neutralization, via a suitable evaporator, e.g. B. falling film evaporator, thin film evaporator or rotary evaporator.
• a suitable evaporator e.g. B. falling film evaporator, thin film evaporator or rotary evaporator.
• Such evaporators are e.g. B. by the companies GIG (4800 Attnang Puchheim, Austria), GEA Canzler (52303 Düren, Germany), Diessel (31103 Hildesheim, Germany) and Pitton (35274 Kirchhain, Germany).
• an additional filtration step can be carried out, in which some activated carbon is added to the solutions or suspensions obtained during the process and the resulting suspension is then filtered. Or the while solutions obtained from the fermentation can be passed over a small activated carbon bed.
• the amounts of activated carbon required for this are in the range of a few% by weight of the solution and are within the knowledge and judgment of the person skilled in the art.
• These filtrations can be facilitated by adding a commercially available flocculant (e.g. Sedipur CF 902 or Sedipur CL 930 from BASF AG, Ludwigshafen) to the respective solution or suspension prior to filtration.
• a commercially available flocculant e.g. Sedipur CF 902 or Sedipur CL 930 from BASF AG, Ludwigshafen
• the fermentation discharge (fermentation broth) is sterilized by heating and then freed from the cell mass by centrifuging, filtering or decanting. After adding 50-1000 mg / kg, preferably 100-200 mg / kg, of a commercially available flocculant based on the fermentation output, the mixture is filtered through a short bed of activated carbon and sand in order to obtain a biomass-free solution with a high D-pantothenic acid content , This prepared solution is then conveyed through the ion exchange bed.
• the processing step according to the invention is not preceded by a separation of the biomass via an anion exchanger
• the fermentation solution containing biomass can advantageously also be passed through the ion exchanger bed from bottom to top, ie against gravity. This procedure is generally advantageous if suspended matter is present in the solution or suspension to be cleaned.
• This solution or suspension can subsequently be dried, for example by spray drying. This can be done in cocurrent, countercurrent or mixed flow. All known atomizers can be used for atomization, in particular centrifugal atomizers (atomizer disks), single-substance nozzles or two-substance nozzles. preferred
• Drying temperature conditions are 150 - 250 ° C tower inlet temperature and 70 - 130 ° C tower outlet temperature. However, it can also be dried at a higher or lower temperature level. In order to achieve a very low residual moisture, a further drying step can be carried out in a fluidized bed. Spray drying can also be carried out in an FSD or SBD dryer (FSD: Fluidized Spray Dryer; SBD: Spray Bed Dryer), as built by the companies Niro (Copenhagen, Denmark) and APV-Anhydro (Copenhagen, Denmark), perform, which are a combination of spray dryer and fluidized bed.
• FSD Fluidized Spray Dryer
• SBD Spray Bed Dryer
• a flow aid can be added during spray drying.
• the deposits on the dryer wall can be reduced and the flow behavior, especially with fine-grained powders, improved.
• Silicates, stearates, phosphates and corn starch are particularly suitable as flow aids.
• drying can also take place in a spray fluidized bed, which can be operated both continuously and batchwise.
• the solution or suspension can be sprayed in from above (top spray), from below (bottom spray) and from the side (sidespray).
• the present invention furthermore relates to a composition for use as an animal feed additive and / or animal feed supplement, it being producible by a) using at least one organism producing D-pantothenic acid, the pantothenic acid (pan) and / or isoleucine / valine (ilv) - biosynthesis is deregulated and which contains at least 2 g / l of salts of D-
• pantothenic acid by fermentation in a culture medium, 0-20 g / l, preferably 0 g / l, of free .beta.-alanine and / or .beta.-alanine salt being added to the culture medium, b) the fermentation solution containing D-pantothenate is passed over an anion exchanger is, c) the D-pantothenate bound to the anion exchanger with a solution containing inorganic or organic calcium and / or magnesium salts in the form of calcium and / or magnesium D-pantothenate or with an HCl solution in the form of free D -Pantothenic acid is eluted, d) optionally the eluate containing free D-pantothenic acid by the addition of
• Calcium and / or magnesium base is adjusted to a pH of 3-10, whereby a solution is obtained which contains calcium and / or magnesium pantothenate and e) the eluate or the solution containing calcium and / or magnesium pantothenate is subjected to drying and / or formulation.
• an elution solution can be used in step c) which contains acidic inorganic and / or organic calcium and / or magnesium salts and / or basic inorganic and / or organic calcium and / or magnesium salts.
• a suspension which contains calcium and / or magnesium pantothenate and which results in the composition according to the invention after further processing can be obtained or presented in step d) or in step e).
• the composition is characterized in that it contains salts of D-pantothenic acid in a concentration of at least 1-100% by weight, preferably at least 20-100% by weight and particularly preferably at least 50% by weight.
• the present invention relates to a composition which contains salts of D-pantothenic acid in the form of divalent cations, preferably calcium and / or magnesium D-pantothenate.
• a composition is preferred which is characterized in that the content of salts of D-pantothenic acid in the form of monovalent cations is ⁇ 1 g / kg.
• a calcium and / or magnesium D-pantothenate is obtained by the process described above, which meets the requirements for a feed additive. These requirements are, for example, a relatively high content of D-pantothenate and good tolerance for the target organism as well as a biological value in the sense of the "vitamin effect" of the product according to the invention.
• the present invention is explained in more detail by the following examples, which, however, are not limiting for the invention:
• aqueous fermentation medium with the following composition is placed:
• the trace salt solution is composed as follows
• 0.15 g Na 2 MoO 4 x 2 H 2 O, 2.5 g H 3 BO 3 , 0.7 g CoCI 2 x 6 H 2 O, 0.25 g CuSO 4 x 5 H 2 O, 1, 6 g MnCI 2 x 4 H 2 O, 0.3 g ZnSO 4 x 7 H 2 O are made up to 1 I with water.
• the trace salt solution is added via sterile filtration.
• the Initial liquid volume is 6 I. The above-mentioned contents are based on this value.
• the fermentation is carried out glucose-limited.
• the pH value is adjusted to 7.2 by adding 25% ammonia solution or 20% phosphoric acid.
• Ammonia also serves as a nitrogen source for the fermentation.
• the speed of the agitator is regulated by keeping the dissolved oxygen content at 30% of the saturation value.
• the fermentation is continued until the dissolved oxygen content (pO 2 ) has reached a value of 95% of the saturation value.
• the fermentation is then ended and the organism thermally killed. For this, the fermentation solution is sterilized for 45 minutes. Successful killing is demonstrated by plating.
• the cells are then separated by centrifugation. After cell separation, the concentration of D-pantothenate is 22.8 g / l after 48 h.
• Fermentation broths with ß-alanine feed-free pantothenic acid titers of over 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, and> can also be produced in an analogous manner
• the same pumping rate is used to rinse with about 1000 ml of demineralized water
• This solution contains cations in the form of NH4 + , Ca 2+ , K + , Mg 2+ and nations, followed by about 1000 ml a 10% calcium chloride solution which elutes D-pantothenic acid bound to the ion exchanger
• the eluate contains pantothenic acid in the form of calcium D-pantothenate, sulfate and phosphate ions of different valences.
• a 75 ml sample of the calcium D-pantothenate solution thus obtained was then dried by evaporating the water on a rotary evaporator and 4.8 g of a light brownish calcium D-pantothenate powder was obtained, which has a content of 35% calcium D-pantothenate. This powder is not prone to sticking and has good product properties.
• aqueous fermentation medium with the following composition is placed:
• the trace salt solution is composed as follows:
• 0.15 g Na 2 MoO 4 x 2 H 2 O, 2.5 g H 3 BO 3 , 0.7 g CoCI 2 x 6 H 2 O, 0.25 g CuSO 4 x 5 H 2 O, 1.6 g MnCI 2 x 4 H 2 O, 0.3 g ZnSO x 7 H 2 O are made up to 1 I with water.
• the trace salt solution is added via sterile filtration.
• the initial liquid volume is 5.5 I. The contents listed above are based on this value.
• the fermentation is carried out glucose-limited.
• the pH was regulated to a value of approximately 7.2 by metering in 25% ammonia solution or 20% phosphoric acid.
• Ammonia also serves as a nitrogen source for the fermentation.
• the speed of the agitator is regulated by keeping the dissolved oxygen content at 30% of the saturation value.
• the fermentation is continued until the dissolved oxygen content (pO 2 ) has reached a value of 95% of the saturation value.
• the fermentation is then ended and the organism thermally killed.
• the fermentation solution is sterilized for 30 minutes. Successful killing is demonstrated by plating.
• the cells are then separated by centrifugation. After cell separation, the concentration of D-pantothenate when terminated after 48 h is 24.1 g / l.
• Fermentation broths with ß-alanine feed-free pantothenic acid titers of over 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, and> can also be produced in an analogous manner Have 90 g / L.
• 1300 ml of the fermenter discharge were conveyed through a bed (volume about 1 liter) of the Lewatit MP 500 ion exchanger (in the Cl form) and rinsed with about 1 liter of water.
• the flow is regulated to approx. 20 ml / min. 2 l of a 5% lime milk suspension were pumped through the ion exchanger at the same flow rate.
• a solution was eluted which contained a calcium D-pantothenate content of 28 g / l. Cations in the form of ammonium, potassium, magnesium or sodium ions were not detected.
• the phosphate ion concentration was reduced by 20% compared to the starting solution upstream of the ion exchanger.
• This aqueous calcium D-pantothenate solution was dried in a laboratory spray dryer from Niro, type Minor.
• the tower inlet temperature was approximately 200 ° C, the tower outlet temperature 85 - 90 ° C.
• the atomization was carried out using a two-fluid nozzle at a pressure of 4 bar. Powder was not added.
• the powdery product had a specification of (data in% by weight): water content: 2 g / 100 g calcium D-pantothenate: 56.3 g / 100 g Ammonium: ⁇ 0.01 g / 100 g of potassium: ⁇ 0.01 g / 100 g of sodium: ⁇ 0.01 g / 100 g of magnesium: ⁇ 0.01 g / 100 g
• a water fermentation medium with the following composition is placed in a laboratory fermenter with stirrer and gassing device with a capacity of 300 l:
• the trace salt solution is composed as follows 0.15 g Na 2 MoO x 2 H 2 O, 2.5 g H 3 BO 3 , 0.7 g CoCI 2 x 6 H 2 O, 0.25 g CuSO 4 x 5 H 2 O, 1, 6 g MnCI 2 x 4 H 2 O, 0.3 g ZnSO 4 x 7 H 2 O are made up to 1 I with water.
• the trace salt solution is added via sterile filtration.
• the initial liquid volume is 100 I. The above contents are based on this value.
• the fermentation is carried out glucose-limited.
• the pH at 7.2 is regulated by adding 25% ammonia solution or 20% phosphoric acid.
• Ammonia also serves as a nitrogen source for the fermentation.
• the speed of the agitator is regulated by keeping the dissolved oxygen content at 30% of the saturation value.
• the fermentation is continued until the dissolved oxygen content (pO 2 ) has reached a value of 95% of the saturation value.
• the fermentation is ended after 43 h.
• the cells are separated by separation in a plate centrifuge. Then the cell-free Fermentation solution sterilized for 60 min. Successful killing is demonstrated by plating.
• the concentration of D-pantothenate after cell separation and sterilization is 21 g / l.
• Fermentation broths with ß-alanine feed-free pantothenic acid titers of over 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, and> can also be produced in an analogous manner Have 90 g / L.
• the fermentation solution was filtered.
• the spray drying was carried out in a laboratory spray dryer from Niro, type Minor.
• the tower inlet temperature averaged about 185 ° C
• Solution 1 was prepared as follows: 87.5 g glucose, 0.5 g CaCl 2 x 2 H 2 O and 5 g MgCl 2 x 6 H 2 O were dissolved in 500 ml demineralized water and sterilized.
• Solution 2 was prepared as follows: 25 ml citrate iron solution (200 g / l sodium citrate, 2 g / L FeSO x 7 H2O, sterile filtered) were mixed with 5 ml trace salt solution (0.15 g Na 2 MoO x 2 H 2 O, 2.5 g H 3 BO 3 , 0.7 g CoCI 2 x 6 H 2 O, 0.25 g CuSO 4 x 5 H 2 O, 1.6 g MnCI 2 x 4 H 2 O, 0.3 g ZnSO x 7 H 2 O were made up to 1L with water filled and sterile filtered) added.
• Solution 3 was prepared as follows: 12.5 g of glucose were made up to 100 ml with water and sterilized.
• Solution 4 was prepared as follows: 25 g of KH 2 PO 4 , 50 g of K 2 HPO, 25 g of NaH 2 PO 4 and 50 g of Na 2 HPO 4 were made up to 500 ml with water and sterilized.
• the mixed medium (volume after addition of all solutions: 5L) was added 100 ml vaccine culture (in SVY medium (SVY medium: Difco Veal Infusion broth 25 g, Difco Yeast extract 5 g, sodium glutamate 5 g, (NH) 2 SO 4 2.7 g were dissolved in 740 ml H 2 O and sterilized; 200 ml sterile 1 MK 2 HPO 4 (pH 7) and 60 ml sterile 50% glucose solution were added (final volume 1L))) of Bacillus subtilis PA668-2A and added at 43 ° C fermented with vigorous stirring at a gassing rate of 12L / min.
• This strain is in accordance with Appendix US serial no. 60 / 262,995.
• a sterile aqueous glucose solution were metered in within 48 h.
• the solution was prepared as follows: 5 kg of glucose x H 2 O and 3.3 g of CaCl 2 x 2 H 2 O were mixed with 2.1 kg of deionized water and sterilized for 30 minutes. Then 11 ml of trace salt solution (composition see above) and 55mL citrate iron solution (composition see above) added. Then 73 ml of sterile-filtered 375 g / L sodium glutamate solution were added.
• the fermentation was carried out glucose-limited. During the fermentation, the pH was kept at 7.2 by adding 25% ammonia solution or 20% phosphoric acid. Ammonia also serves as a nitrogen source for the fermentation. The speed of the stirrer was controlled by keeping the dissolved oxygen content at 20% of the saturation value. The foaming was controlled by occasionally adding the anti-foaming agent Tego KS 911. After the addition of the carbon source had been discontinued, the fermentation was continued until the dissolved oxygen content (pO 2 ) had reached a value of 95% of the saturation value. The fermentation was then ended. The cells were separated by centrifugation. The remaining cells in the supernatant were thermally killed by sterilization. The killing was verified by plating.
• Fermentation broths with a ß-alanine-free feed pantothenic acid titer of more than 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 can also be produced in an analogous manner , and have more than 90 g / L.
• the eluate contained pantothenic acid in the form of calcium D-pantothenate, chloride, sulfate, and phosphate ions of different valences.
• This aqueous calcium D-pantothenate solution was dried in a laboratory spray dryer from Niro, type Minor.
• the tower inlet temperature was approximately 200 ° C, the tower outlet temperature 85 - 90 ° C.
• the atomization was carried out using a two-fluid nozzle at a pressure of 4 bar. Powder was not added.
• the powdery product had a specification of (data in% by weight): Water content: 2% D-pantothenate: 24% ammonium: 0.05% potassium: 0.09% sodium: 0.02%
• Ion exchanger has been reduced.
• Example 7 1500 ml of the filtrate obtained from Example 4 were conveyed through a bed (1 liter) of the Lewatit MP500 ion exchanger (in the OH " form). It was rinsed with water. The flow rate was approximately 20 ml / min. About two liters of an approximately 5% hydrochloric acid solution were passed over the ion exchanger at the same flow rate, and the eluate was collected and adjusted to a pH of approximately 7 with 20% milk of lime (suspension of potassium hydroxide in water) filtered again through a paper filter and obtained as the filtrate 2174 g of an aqueous calcium D-pantothenate solution with a D-pantothenate content of 19 g / l.
• a 75 ml sample of the calcium D-pantothenate solution thus obtained was then dried by evaporating the water on a rotary evaporator and 5.7 g of a brownish calcium D-pantothenate powder was obtained, which had a content of 25% D-pantothenate. This powder is not prone to sticking and has good product properties.
• aqueous calcium D-pantothenate solution has a D-pantothenate content of 21 g / l.
• a 75 ml sample of the calcium D-pantothenate solution thus obtained is then dried by evaporating the water on a rotary evaporator and 5.6 g of a brownish calcium D-pantothenate powder is obtained, which has a content of 28% D-pantothenate. This powder is not prone to sticking and has good product properties.

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